Field of the Invention
The present invention relates to a method and a kit for amplifying a target DNA sequence and producing concatemers. The present invention further relates to a method, a kit, and an apparatus for determining a nucleotide sequence using the thus produced concatemers.
Background Art
In recent years, a rapid and highly sensitive nucleotide sequencing method based on massively parallel nucleotide sequencing has been developed (Non-patent Document 1), and the widespread use of apparatuses involving such technology makes it possible to analyze the full genome of a plant, a fungus, an animal, a bacterium, or a virus within 1 week. The obtained nucleotide sequence information is now crucial in the fields of drug discovery, medicine, and agriculture. The range of the applications of genetic sequence information will undoubtedly further expand. Further improvement in throughput and accuracy will be required in the future. Moreover, it is also considered that fields such as the field of expression analysis requiring accurate quantitative performance will experience significant growth.
In massively parallel nucleotide sequencing, millions to billions of monoclonal DNA fragment clusters are disposed on a flow path substrate, and then the nucleotide sequences of DNA fragments of each cluster are read in parallel, thereby realizing a high throughput. The means employed for the production of many clusters and the disposition of the clusters on a flow path substrate are techniques such as (a) PCR that is performed with an end of template DNA immobilized on a flow path substrate, (b) immobilization of emulsion PCR (emPCR) products to solid beads, and (c) formation of DNA nanoballs by isothermal amplification using cyclic DNA.